Method for promoting cell proliferation and growth

ABSTRACT

A method is disclosed which provides for the proliferation and growth of cells, such as prostate cells, by contacting them with an effective amount of a placental-derived prostate growth factor.

This application is a division of application Ser. No. 08/735,041, filedOct. 22, 1996, now U.S. Pat. No. 5,914,251.

FIELD OF THE INVENTION

This invention relates in general to polypeptides with growth promotingactivities. More specifically, the invention concerns: growth factors(herein termed placental-derived prostate factors, or "PDPFs"), havingthe ability to stimulate the proliferation and growth of prostate cellsand other cells; recombinant methods for the production of such growthfactors; methods for the use of such growth factors to stimulate cellproliferation, growth and/or survival; diagnostic methods for thedetection of such factors in biological samples by the use of PDPFantibodies and labeled nucleic acid probes; and methods for theidentification of inhibitors against PDPF that will be potentiallyuseful as anti-cancer agents.

BACKGROUND OF THE INVENTION

It has been estimated that each year, nearly two hundred and fiftythousand men in the United States are diagnosed with prostate cancer andthat approximately forty thousand men will die from it; Boring et al.,CA Cancer Journal for Clinicians, Volume 44, Number 1, pages 7-26(1994). Current treatments, which include surgery, radiation and hormoneablation, have some effect on slowing tumor growth but show nosignificant effect on long term remission or cure; Scher, CurrentOpinion in Oncology, Volume 3, pages 568-574 (1991). When confinedwithin the organ capsule, prostate carcinoma is relatively easy to treatsuccessfully. However, metastatic disease, which targets to the axialskeleton about eighty percent of the time, is nearly always refractoryto treatment; Franks, Journal of Pathology and Bacteriology, Volume 72,pages 603-611 (1956); and Huben et al., CA Cancer Journal forClinicians., Volume 36, Number 5, pages 274-292 (1986).

The dissemination of prostate carcinoma cells to the bones of thecentral spine has been ascribed to the presence of paravertebralvessels; Zetter et al., Prostate Cancer and Bone Metastasis, edited byKarr and Yamanaka, Plenum Press, New York (1992), at pages 39-43.However, this direct metastatic route does not explain why the level oftumor cell proliferation in bone is often higher than in the prostategland itself; Jacobs, Urology, Volume 21, Number 4, pages 337-344(1983). Additionally, bone metastases are often the first evidence ofprogression to androgen-independent prostate carcinoma; Logothetis etal., Seminars in Oncology, Volume 21, Number 5, pages 620-629 (1994).The uncoupling of prostate carcinoma cell growth from hormoneresponsiveness signals the failure or irrelevance of hormone ablationtherapy and the increasing ability of prostate carcinoma cells toproliferate in response to paracrine and autocrine peptide growthfactors; Thompson, Cancer Cells, Volume 2, Number 11, pages 345-354(1990); and Scher et al., Seminars in Urology, Volume 10, Number 1,pages 55-64 (1992). Although clinical relevance has not beenconvincingly established, several growth factors, including bFGF, aFGF,EGF, TGFa, and PDGF, have been isolated from the conditioned media ofhuman prostate carcinoma cells in vitro, and from prostate tissue invivo.

Bone stromal cells also produce factors that stimulate the proliferationof prostate carcinoma cells; Chackal-Roy et al., Journal of ClinicalInvestigation, Volume 84, pages 43-50 (1989). The interactions ofprostate stromal and epithelial cells with regard to KGF and bFGF havebeen well established; Chung, Cancer Biology, Volume 4, pages 183-192(1993). However, the interactions between bone stromal cells andmetastatic prostate carcinoma cells has not yet been studied as closely.Conditioned media from bone stromal cells, but not skeletal muscle,keratinocytes or kidney epithelial cells, stimulates the proliferationof prostate carcinoma cells; see Zetter et al. above. Furthermore, bonestromal cell media is not active on kidney carcinoma or melanoma cells,suggesting tissue specific interactions between bone stromal cells andprostate carcinoma cells. This tissue specificity has been confirmed invivo by co-inoculating stromal cells and LNCaP prostate carcinoma cells,which are normally non-tumorigenic, into nude mice; Gleave et al.,Cancer Research, Volume 51, pages 3753-3761 (1991). In these studies,bone or prostate stromal cells supported tumor growth in vivo, whereaslung or kidney stromal cells did not.

One factor, identified in bone stromal cell cultures, that stimulatesprostate cell proliferation is transferrin, which is awell-characterized protein involved in the transport of iron across cellmembranes; Rossi et al., Proceedings of the National Academy of ScienceU.S.A., Volume 89, pages 6197-6201 (1992). In addition, published PCTpatent application PCT/US95/09261 (WO 96/04379) describes the isolationand expression of DNA encoding a bone and prostate-derived growthfactor, termed BPGF-1. The BPGF-1 gene was found to be expressedpredominantly in bone, prostate tissue and seminal vesicles, and BPGF-1polypeptide stimulated the proliferation of prostatic epithelial cellsin vitro.

SUMMARY OF THE INVENTION

The present invention provides novel growth factors which stimulate thegrowth of prostate and other cell types, and which are distinct fromBPGF-1. The growth factors of this invention are polypeptides (i.e.,proteins) that stimulate the proliferation of several prostate carcinomacell lines, but not normal prostate epithelial cells. In addition, thegrowth of other tumor cells derived from placenta, oral epithelium,palate and bone is also stimulated. The growth factors of this inventionare herein individually and collectively termed "placenta-derivedprostate factor", or "PDPF", based on the tissue origin of the cDNAlibrary used to clone the encoding nucleic acid molecules and the factthat these factors have activity against prostate cells. Of two variablyspliced forms specifically identified so far, the shorter polypeptideform (termed PDPF-1) appears to be more active in the cell growth assaysevaluated.

The present invention also encompasses genes and nucleic acid moleculesthat encode such polypeptides, as well as recombinant methods for theuse of the genes and nucleic acid molecules to produce the polypeptides.Additionally, this invention is directed to the use of the polypeptidesto promote the growth of prostate and other cells responsive to thegrowth promoting effects of the polypeptides, either in vitro or invivo. The protein growth factors of this invention can also be used inassays and screens for the identification of antagonists, i.e.,inhibitors of PDPF cell proliferation stimulating activity, suchantagonists being potentially useful as anti-tumor agents for thetreatment of responsive tumors. The invention is further concerned withthe use of PDPF antibodies and nucleic acid molecules encoding PDPF, orfragments (i.e., segments) thereof, in diagnostic methods for thedetection of PDPF in biological samples such as tissue or fluid samples,including their use in diagnostic methods for the detection of tumors.

Especially preferred embodiments of this invention are nucleic acidmolecules having the DNA sequences of SEQ ID NOS: 3 or 5, and proteinshaving the amino acid sequences of SEQ ID NOS: 4 or 6. Also preferred isan alternative nucleic acid sequence to the one of SEQ ID NO: 5, inwhich the base "C" at position 1260 is changed to "G" (but otherwiseencoding the same polypeptide) (SEQ ID NO: 7). In addition to thesepreferred embodiments, the invention also encompasses fragments (such astruncation analogs or interior segments of the proteins) and otherderivative molecules of modified sequence, including substitution,deletion and addition analogs, having the growth promoting activity ofthe full length sequence of PDPF, as will be more fully described in thefollowing text.

The terms "gene", "DNA", and "nucleic acid molecule" used herein withreference to this invention are meant to refer to isolated moleculesthat are free of total genomic DNA of a particular species, whileretaining coding sequences for the described polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. These figures show the DNA sequence (SEQ ID NO: 1) andpredicted amino acid sequence (SEQ ID NO: 2) of the polypeptide known asBPGF-1, as disclosed in the above mentioned WO 96/04379.

FIGS. 2A-2C. These figures show the DNA sequence (SEQ ID NO: 3) andpredicted amino acid sequence (SEQ ID NO: 4) of a growth promotingpolypeptide (PDPF-1) in accordance with the present invention.

FIGS. 3A-3D. These figures show the DNA sequence (SEQ ID NO: 5) andpredicted amino acid sequence (SEQ ID NO: 6) of another growth promotingpolypeptide (PDPF-2) in accordance with the present invention.

FIGS. 4A-4G. These figures show the results of an analysis of varioustissue sections taken from normal embryonic and adult rats, using forthe analysis a DNA probe for PDPF mRNA that had been radiolabeled with³³ P. As shown in the figure, many structures in the embryo (FIG. 4A)express mRNA for PDPF, including kidney, pancreas, heart and skin. Thereis also strong signal in the chorionic plate and the maternal tissue ofthe placenta. In the adult male reproductive tract, the epididymalepithelia (middle row, FIG. 4B) express mRNA for PDPF, the seminiferoustubules (middle row, FIG. 4C) express varying amounts of mRNA for PDPF,and the vesicular gland epithelia (middle row, FIG. 4D) demonstrated thehighest level of expression of mRNA for PDPF. Bright field images of themiddle row panels are adjacent on the right. (FIGS. 4E, 4F, and 4G).

FIG. 5. This figure shows the expression of PDPF in prostate carcinomacells and normal prostate epithelial cells. Conditioned media (CM) wascollected and assayed by Western blot using a PDPF-specific antibody.Lane 1, recombinant form of protein PDPF-1 (SEQ ID NO: 4). Lane 2,recombinant form of protein PDPF-2 (SEQ ID NO: 6). Lane 3, CM fromuntransfected cells. Lane 4, CM from LNCaP prostate carcinoma cells.Lane 5, CM from DU145 prostate carcinoma cells. Lane 6, CM from PC3prostate carcinoma cells. Lane 7, CM from NbE1.4 normal prostateepithelial cells.

DETAILED DESCRIPTION OF THE INVENTION

In addition to the polypeptides of FIGS. 2A-2C (SEQ ID NO: 4) and FIGS.2A-2C and 3A-3D (SEQ ID NO: 6), also intended as part of this inventionare fragments, precursors and derivatives thereof that are substantiallybiologically equivalent. By "substantially biologically equivalent" ismeant having the same properties of the polypeptides as describedherein, even if in lesser or greater degree. Preferably, such analogswill cross-react with antibodies raised against the polypeptides ofFIGS. 2A-2C and 3A-3D. The term "derivative" is intended to meanmolecules representing one or more amino acid substitutions, deletionsand/or additions derived from the linear array of amino acids of suchpolypeptides, and which are also substantially biologically equivalentor share one or more biological properties.

Especially preferred derivatives in accordance with this invention arethose which possess a degree of homology (i.e., identity of amino acidresidues) with the proteins of FIGS. 2A-2C (SEQ ID NO: 4) or FIGS. 3A-3D(SEQ ID NO: 6) in excess of eighty percent (80%), and most preferably,in excess of ninety percent (90%), excluding the amino acid sequence ofBPGF-1, above, which is outside the scope of the present invention.

Percent sequence identity can be determined by standard methods that arecommonly used to compare the similarity in position of the amino acidsof two polypeptides, to generate an optimal alignment of two respectivesequences. By way of illustration, using a computer program such asBLAST or FASTA (FASTA is preferred for the present invention), twopolypeptides are aligned for optimal matching of their respective aminoacids (either along the full length of one or both sequences, or along apre-determined portion of one or both sequences). The programs provide a"default" opening penalty and a "default" gap penalty, and a scoringmatrix such as PAM 250. A standard scoring matrix can be used inconjunction with the computer program; see Dayhoff et al., in Atlas ofProtein Sequence and Structure, Volume 5, Supplement 3 (1978). Thepercent identity (or "homology" as used herein) can then be calculatedas follows: ##EQU1##

Polypeptides in accordance with this invention that are at least eightypercent (80%) identical, as determined by the above method, willtypically have one or more amino acid substitutions, deletions, and/orinsertions. Usually, the substitutions will be conservative so as tohave little or no effect on the overall net charge, polarity, orhydrophobicity of the protein. Examples of conservative substitutionsare set forth below.

    ______________________________________                                        Conservative amino acid substitutions                                         ______________________________________                                        Basic:              arginine                                                                      lysine                                                                        histidine                                                 Acidic:             glutamic acid                                                                 aspartic acid                                             Polar:              glutamine                                                                     asparagine                                                Hydrophobic:        leucine                                                                       isoleucine                                                                    valine                                                    Aromatic:           phenylalanine                                                                 tryptophan                                                                    tyrosine                                                  Small:              glycine                                                                       alanine                                                                       serine                                                                        threonine                                                                     methionine                                                ______________________________________                                    

PDPF polypeptides of this invention may or may not have an aminoterminal methionine, depending on the manner in which they are prepared.Typically, an amino terminal methionine residue will be present when thepolypeptide is produced recombinantly in a non-secreting bacterial(e.g., E. coli) strain as the host.

PDPF fragments included within this invention will be those that haveless than the full length sequence, but which possess substantially thesame biological activity and are truncated at the amino terminus, thecarboxy terminus, and/or internally.

This invention also encompasses nucleic acid molecules encoding any ofthe above mentioned polypeptides. Besides the nucleic acid moleculeshaving the nucleotide sequences shown in FIGS. 2A-2C (SEQ ID NO: 3) andFIGS. 3A-3D (SEQ ID NO: 5), also included are degenerate sequencesthereof encoding the same polypeptides. In addition, this inventionencompasses nucleic acid molecules encoding biologically activeprecursors, fragments and derivatives of the polypeptides describedherein. In addition, the invention encompasses nucleic acid moleculesencoding the complementary (i.e., antisense) strands, as well as DNAmolecules which hybridize (or would hybridize but for the variability ofnucleotide sequence due to the degeneracy of codons) to the DNAmolecules of FIGS. 2A-2C and 3A-3D, to fragments or degenerate sequencesthereof, or to their complementary strands, preferably under relativelystringent conditions (e.g., conditions such as described below).Excluded from this group is the nucleic acid molecule encoding BPGF-1.The present invention also embraces nucleic acid molecules that mayencode additional amino acid residues flanking the 5' or 3' portions ofthe region encoding the "mature" PDPF polypeptide (i.e., the processedproduct harvested from the host), such as sequences encoding alternativepre/pro regions (i.e., sequences responsible for secretion of thepolypeptide through cell membranes) in place of the "native" pre/proregions. The additional sequences may also be noncoding sequences,including regulatory sequences such as promoters of transcription ortranslation, depending on the host cell. The nucleic acid molecules mayeven include various internal non-coding sequences (i.e., introns) knownto occur within genes.

As mentioned, the present invention contains within its scope DNAmolecules which are hybridizable to complementary sequences underrelatively stringent conditions. In general, as employed herein the term"stringent conditions" refers to hybridization and washing underconditions that permit the binding of a nucleic acid molecule such as anoligonucleotide or cDNA molecule to highly homologous sequences.

One stringent washing solution, suitable for use with cDNA probes at atemperature of 55-65° C., is composed of 0.015 M sodium chloride, 0.0015M sodium citrate (0.1×SSC, where 1×SSC=0.15 M sodium chloride and 0.015M sodium citrate) and 0.1 percent SDS. Another, slightly less stringentwashing solution, is composed of 0.2×SSC and 0.1 percent SDS, and can beused at a temperature of between 50-65° C.

Where oligonucleotide probes are used to screen cDNA or genomiclibraries, the following stringent washing conditions may be used. Oneprotocol uses 6×SSC with 0.05 percent sodium pyrophosphate at atemperature of 35-62° C., depending on the length of the oligonucleotideprobe. For example, 14-base pair probes are washed at 35-40° C., 17-basepair probes are washed at 45-50° C., 20-base pair probes are washed at52-57° C., and 23-base pair probes are washed at 57-63° C. Thetemperature can be increased 2-6° C. where background non-specificbinding appears to be high. Another protocol utilizestetramethylammonium chloride (TMAC) for washing oligonucleotide probes.A suitable stringent washing solution is composed of 3 M TMAC, 50 mMTris-HCl, pH 8.0, and 0.2 percent SDS. The washing temperature for usewith this solution is a function of the length of the probe. Forexample, a 17-base pair probe is typically washed at about 45-50° C.

Also included within the scope of this invention are RNA molecules thatare homologous to any of the aforementioned DNA molecules.

Recombinant Methods of Preparation

The full length polypeptides of the invention, or fragments orderivatives thereof, can be prepared using well known recombinant DNAtechnology methods such as those set forth in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989) and/or Ausubel et al., Editors, CurrentProtocols in Molecular Biology, Green Publishers Inc. and Wiley andSons, New York (1994). A gene or cDNA encoding the polypeptide ortruncated version thereof may be obtained, for example, by screening agenomic or cDNA library, or by PCR amplification. Alternatively, a geneencoding the polypeptide or fragment, or analog thereof, may be preparedby chemical synthesis using methods well known to the skilled artisan,such as those described by Engels et al. in Angew. Chem. Intl. Ed.,Volume 28, pages 716-734 (1989). Typically, the DNA encoding thepolypeptide will be several hundred nucleotides in length. Nucleic acidslarger than about one hundred nucleotides can be synthesized as severalfragments using these same methods and the fragments can then be ligatedtogether to form a nucleotide sequence of the desired length.

The nucleic acid analogs may be produced using site directed mutagenesisor PCR amplification where the primer(s) have the desired pointmutations (see Sambrook et al., above, and Ausubel et al., above, fordescriptions of suitable mutagenesis techniques). Chemical synthesisusing methods described by Engels et al., above, may also be used toprepare such variants. Preferred nucleic acid analogs are thosecontaining nucleotide substitutions accounting for codon preference inthe host cell to be used to produce the polypeptide. Other preferredvariants are those encoding conservative amino acid changes (see above)as compared to wild type, and/or those designed to either generate ordelete glycosylation and/or phosphorylation site(s) on the polypeptide.

The genes or cDNAs of this invention can be inserted into an appropriateexpression vector for expression in a suitable host cell. The vector isselected to be functional in the particular host cell employed (i.e.,the vector is compatible with the host cell machinery, such thatamplification and/or expression of the gene can occur). The polypeptide,or fragment or derivative analog thereof, may be amplified/expressed inprokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic hostcells. Selection of the host cell will depend at least in part onwhether the polypeptide expression product is to be glycosylated. Ifglycosylation is desired, then yeast, insect or mammalian host cells arepreferred for use, in that yeast cells will glycosylate the polypeptide,and insect and mammalian cells can glycosylate and/or phosphorylate thepolypeptide in a manner similar to "native" glycosylation and/orphosphorylation.

The vectors used in any of the host cells to express the polypeptide mayalso contain a 5' flanking sequence (also referred to as a "promoter")and other expression regulatory elements operatively linked to the DNAto be expressed, as well as enhancer(s), an origin of replicationelement, a transcriptional termination element, a complete intronsequence containing a donor and acceptor splice site, a signal peptidesequence, a ribosome binding site element, a polyadenylation sequence, apolylinker region for inserting the nucleic acid encoding thepolypeptide to be expressed, and a selectable marker element. Each ofthese elements is discussed below.

Optionally, the vector may also contain a "tag" sequence, i.e., anoligonucleotide sequence located at the 5' or 3' end of thepolypeptide-coding sequence that encodes polyHis (such as hexaHis) oranother small immunogenic sequence. This tag will be expressed alongwith the protein, and can serve as an affinity tag for purification ofthe polypeptide from the host cell. Optionally, the tag can subsequentlybe removed from the purified polypeptide by various means, for example,with use of a selective peptidase.

The 5' flanking sequence may be the native 5' flanking sequence, or itmay be homologous (i.e., from the same species and/or strain as the hostcell), heterologous (i.e., from a species other than the host cellspecies or strain), hybrid (i.e., a combination of 5' flanking sequencesfrom more than one source), or synthetic. The source of the 5' flankingsequence may be any unicellular prokaryotic or eukaryotic organism, anyvertebrate or invertebrate organism, or any plant, provided that the 5'flanking sequence is functional in, and can be activated by the hostcell machinery.

Where the 5' flanking sequence is not known, a fragment of DNAcontaining a 5' flanking sequence may be isolated from a larger piece ofDNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion using one or more carefully selected enzymes to isolate theproper DNA fragment. After digestion, the desired fragment may beisolated by agarose gel purification, or by other methods known to theskilled artisan. Selection of suitable enzymes to accomplish thispurpose will be readily apparent to one skilled in the art.

The origin of replication element is typically a part of prokaryoticexpression vectors purchased commercially, and aids in the amplificationof the vector in a host cell. Amplification of the vector to a certaincopy number can, in some cases, be important for optimal expression ofthe polypeptide. If the vector of choice does not contain an origin ofreplication site, one may be chemically synthesized based on a knownsequence, and then ligated into the vector.

The transcription termination element is typically located 3' to the endof the polypeptide coding sequence and serves to terminate transcriptionof the mRNA. Usually, the transcription termination element inprokaryotic cells is a G-C rich fragment followed by a poly T sequence.While the element is easily cloned from a library or even purchasedcommercially as part of a vector, it can also be readily synthesizedusing methods for nucleic acid synthesis such as those referred toabove.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that: (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline or kanamycin for prokaryotic host cells, and, e.g.,neomycin for mammalian host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex media. Preferred selectable markers for use in prokaryoticexpression are the kanamycin resistance gene, the ampicillin resistancegene, and the tetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarnosequence (for prokaryotes) or the Kozak sequence (for eukaryotes), isnecessary for the initiation of translation of mRNA. The element istypically located 3' to the promoter and 5' to the coding sequence ofthe polypeptide to be synthesized. The Shine-Dalgarno sequence is variedbut is typically a polypurine (i.e., having a high A-G content). ManyShine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector. The Kozak sequence typically includes sequencesimmediately before and after the initiating codon. A preferred Kozaksequence is one that is associated with a high efficiency of initiationof translation at the AUG start codon.

In those cases where it is desirable for the polypeptide to be secretedfrom the host cell, a signal sequence may be used to direct thepolypeptide out of the host cell where it is synthesized. Typically, thesignal sequence is positioned in the coding region of nucleic acidsequence, or directly at the 5' end of the coding region. Many signalsequences have been identified, and any of them that are functional inthe selected host cell may be used here. Consequently, the signalsequence may be homologous or heterologous to the polypeptide.Additionally, the signal sequence may be chemically synthesized usingmethods referred to above.

After the vector has been constructed and a nucleic acid has beeninserted into the proper site of the vector, the completed vector may beinserted into a suitable host cell for amplification and/or polypeptideexpression.

Host cells may be prokaryotic (such as E. coli) or eukaryotic (such as ayeast cell, an insect cell, or a vertebrate cell). The host cell, whencultured under appropriate conditions, can synthesize the polypeptide,which can subsequently be collected from the culture medium (if the hostcell secretes it into the medium) or directly from the host cellproducing it (if it is not secreted). After collection, the polypeptidecan be purified using methods such as molecular sieve chromatography,affinity chromatography, and the like.

Selection of the host cell will depend in part on whether thepolypeptide is to be glycosylated or phosphorylated (in this caseeukaryotic host cells are preferred), and the manner in which the hostcell is able to "fold" the polypeptide into its native tertiarystructure (e.g., proper orientation of disulfide bridges, etc.) suchthat biologically active polypeptide is prepared by the cell. However,where the host cell does not synthesize biologically active polypeptide,it may be "folded" after synthesis using appropriate chemical conditionsas discussed below.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO) or 3T3 cells. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. Other suitable mammalian cell lines are the monkeyCOS-1 and COS-7 cell lines, and the CV-1 cell line. Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Still other suitable mammalian cell lines include butare not limited to, HeLa, mouse L-929 cells, 3T3 lines derived fromSwiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.

Also useful as host cells are bacterial cells. For example, the variousstrains of E. coli (e.g., HB101, DH5a, DH10, and MC1061) are well-knownas host cells in the field of biotechnology. Various strains of B.subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp., andthe like, may also be employed. Additionally, many strains of yeastcells known to those skilled in the art are also available as host cellsfor expression of the polypeptides of the present invention. Also, wheredesired, insect cells may be utilized as host cells. See, for example,Miller et al., Genetic Engineering, Volume 8, pages 277-298 (1986).

Insertion (also referred to as "transformation" or "transfection") ofthe vector into the selected host cell may be accomplished using suchmethods as calcium phosphate, electroporation, microinjection,lipofection or the DEAE-dextran method. The method selected will in partbe a function of the type of host cell to be used. These methods andother suitable methods are well known to the skilled artisan, and areset forth, for example, in Sambrook et al., above.

The host cells containing the vector may be cultured using standardmedia well known to the skilled artisan. The media will usually containall nutrients necessary for the growth and survival of the cells.Suitable media for culturing E. coli cells are, for example, Luria Broth(LB) and/or Terrific Broth (TB). Suitable media for culturing eukaryoticcells are RPMI 1640, MEM, DMEM, all of which may be supplemented withserum and/or growth factors as required by the particular cell linebeing cultured. A suitable medium for insect cultures is Grace's mediumsupplemented with yeastolate, lactalbumin hydrolysate and/or fetal calfserum, as necessary.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of polypeptide produced in the host cell can be evaluatedusing standard methods known in the art. Such methods include, withoutlimitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or activity assays such as DNA binding gelshift assays.

If the polypeptide has been designed to be secreted from the host cells,the majority of polypeptide will likely be found in the cell culturemedium. If the polypeptide is not secreted, it will be present in thecytoplasm (for eukaryotic, gram-positive bacteria, and insect hostcells) or in the periplasm (for gram-negative bacteria host cells).

For intracellular polypeptide, the host cells are typically firstdisrupted mechanically or osmotically to release the cytoplasmiccontents into a buffered solution. The polypeptide is then isolated fromthis solution. Purification of the polypeptide from solution canthereafter be accomplished using a variety of techniques. If thepolypeptide has been synthesized so that it contains a tag such ashexahistidine or other small peptide at either its carboxyl or aminoterminus, it may essentially be purified in a one-step process bypassing the solution through an affinity column where the column matrixhas a high affinity for the tag or for the polypeptide directly (i.e., amonoclonal antibody). For example, polyhistidine binds with greataffinity and specificity to nickel, thus an affinity column of nickel(such as the Qiagen nickel columns) can be used for purification. (See,for example, Ausubel et al., eds., Current Protocols in MolecularBiology, above).

Where, on the other hand, the polypeptide has no tag and no antibodiesare available, other well known procedures for purification can be used.Such procedures include, without limitation, ion exchangechromatography, molecular sieve chromatography, HPLC, native gelelectrophoresis in combination with gel elution, and preparativeisoelectric focusing ("Isoprime" machine/technique, Hoefer Scientific).In some cases, two or more of these techniques may be combined toachieve increased purity.

If it is anticipated that the polypeptide will be found primarily in theperiplasmic space of the bacteria or the cytoplasm of eukaryotic cells,the contents of the periplasm or cytoplasm, including inclusion bodies(e.g., gram-negative bacteria) if the processed polypeptide has formedsuch complexes, can be extracted from the host cell using any standardtechnique known to the skilled artisan. For example, the host cells canbe lysed to release the contents of the periplasm by the use of a Frenchpress, homogenization, and/or sonication. The homogenate can then becentrifuged.

In addition to the use of recombinant DNA techniques, the polypeptides,fragments, and/or derivatives thereof, may be prepared by chemicalsynthesis methods (such as solid phase peptide synthesis) using methodsknown in the art, including those set forth by Merrifield et al. in theJournal of the American Chemical Society, Volume 85, page 2149 (1964),by Houghten et al. in the Proceedings of the National Academy of ScienceU.S.A., Volume 82, page 5132 (1985), and by Stewart and Young in SolidPhase Peptide Synthesis, Pierce Chemical Co, Rockford, Ill. (1984).Chemically synthesized polypeptides or fragments or analogs thereof maybe oxidized using methods set forth in these references to formdisulfide bridges. The resulting polypeptide products may be employed asbiologically active or immunological substitutes for the natural,purified polypeptide.

Chemically modified polypeptide compositions where the polypeptide islinked to a polymer in order to modify properties are included withinthe scope of the present invention. The polymer is typically watersoluble so that the protein to which it is attached does not precipitatein an aqueous environment, such as a physiological environment. Thepolymer may have a single reactive group, such as an active ester foracylation or an aldehyde for alkylation, so that the degree ofpolymerization may be controlled. A preferred reactive aldehyde ispolyethylene glycol propionaldehyde, which is water stable, or monoC1-C10 alkoxy or aryloxy derivatives thereof (see U.S. Pat. No.5,252,714). The polymer may be branched or unbranched. Preferably, fortherapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable. The water soluble polymer, or mixturethereof if desired, may be selected from the group consisting of, forexample, polyethylene glycol (PEG), monomethoxy-polyethylene glycol,dextran, cellulose, or other carbohydrate based polymers, poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol. The polymer preferred forchemical modification is polyethylene glycol.

Pegylation of the polypeptide (i.e., addition of polyethyene glycolchains) may be carried out by any of the pegylation reactions known inthe art, as described for example in the following references: Focus onGrowth Factors, Volume 3, Number 2, pages 4-10 (1992); EP 0 154 316; andEP 0 401 384. Preferably, the pegylation is carried out via an acylationreaction or an alkylation reaction with a reactive polyethylene glycolmolecule (or an analogous reactive water-soluble polymer), in accordancewith known methods.

The polypeptide growth factors of this invention (including fragments,derivatives and the above-described chemically modified productsthereof) can be used alone or together with other components in tissueculture media to promote the growth and proliferation of responsivecells, e.g., prostate cells. Various assay methods can be used to detectand quantitatively measure such cell growth. Such methods include,merely by way of example, in vitro assays such as the uptake and elutionof crystal violet dye; the incorporation of radioactive ornon-radioactive labels, such as ³ H-thymidine or bromodeoxy uridine,into precipitable cellular DNA, the bioreduction of MTS (Owen's reagent)to formazan by dehydrogenase enzymes in metabolically active cells, andthe direct counting of cells following trypsinization and resuspensionin media using a Coulter Counter.

Generation of Antibodies

The polypeptides of the invention, including fragments and/orderivatives thereof, may also be used to generate antibodies inaccordance with standard methods. The antibodies may be polyclonal,monoclonal, recombinant, chimeric, single-chain and/or bispecific, etc.To improve the likelihood of producing an immune response, the aminoacid sequence of the polypeptide can be analyzed to identify portions ofthe molecule that may be associated with increased immunogenicity. Forexample, the amino acid sequence may be subjected to computer analysisto identify surface epitopes, such as in accordance with the method ofHope and Woods, Proceedings of the National Academy of Science U.S.A.,Volume 78, pages 3824-3828 (1981). Alternatively, the deduced amino acidsequence from different species can be compared and relativelynon-homologous regions identified (e.g., sequences of at least about sixcontiguous amino acids in which at least about two amino acids differfrom one species to the other). These non-homologous regions would bemore likely to be immunogenic across various species.

Various procedures known in the art can be used for the production ofpolyclonal antibodies which recognize epitopes of the polypeptides ofthis invention. For the production of antibody, various host animals canbe immunized by injection with the polypeptide, or fragment orderivative thereof, including but not limited to rabbits, mice, rats,etc. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's, mineral gels such as aluminum hydroxide (alum), surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as Bacille Calmette-Guerin andCorynebacterium parvum.

For the preparation of monoclonal antibodies directed toward thepolypeptides, any technique which provides for the production ofantibody molecules by continuous cell lines in culture may be used. Forexample, the hybridoma technique originally developed by Kohler andMilstein which is described in Nature, Volume 256, pages 495-497 (1975),as well as the trioma technique, the human B-cell hybridoma techniquedescribed by Kozbor et al. in Immunology Today, Volume 4, page 72(1983), and the EBV-hybridoma technique to produce monoclonal antibodiesdescribed by Cole et al. in "Monoclonal Antibodies and Cancer Therapy",Alan R. Liss, Inc., pages 77-96 (1985), are all useful for preparationof monoclonal antibodies in accordance with this invention.

In addition, molecular clones of an antibody to an epitope or epitopesof the polypeptides can be prepared with known techniques. Inparticular, recombinant DNA methodology may be used to construct nucleicacid sequences which encode a monoclonal antibody molecule orantigen-binding region thereof; see, for example, Maniatis et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982).

DESCRIPTION OF SPECIFIC EMBODIMENTS

The invention is now described in further detail with reference to thefollowing specific test methods, procedures, and results.

Cloning of PDPF

From the cDNA sequence of BPGF-1 (SEQ ID NO: 1; See WO 96/04379), itcould be seen that the predicted amino acid sequence (SEQ ID NO: 2)encoded by the long open reading frame of this nucleic acid sequence didnot appear to have a signal sequence, as is usually found in a secretedprotein. Consequently, an attempt was made to isolate other clones. Morespecifically, the nucleic acid sequence of SEQ ID NO: 1 was used todesign polymerase chain reaction (PCR) primers to screen tissue cDNAsand also cDNA libraries for other BPGF clones. Initial screening of thefirst strand cDNAs indicated that BPGF is expressed in the humanplacenta. To prepare a cDNA library, cDNA was prepared from humanplacental polyA⁺ RNA using a NotI-oligo(dT) primer and SalI adapter,then size-fractionated, and thereafter cloned into vector pSPORT1(Bethesda Research Laboratories, Bethesda, Md.), using the proceduresprovided by the manufacturer; see D'Alessio et al., Focus, Volume 12,pages 47-48 (1990). Transformants were divided into fifty-two pools ofapproximately six thousand clones each, and plasmid DNA (0.5 mg/L) wasprepared from each pool; Del Sal et al., Biotechniques, Volume 7, pages514-520 (1989). The pooled DNAs were mixed four at a time into thirteenmaster pools of approximately twenty thousand clones each. Approximately100-200 ng of each master pool DNA was used as template in a PCRprocedure carried out with the primers

    5'TGGAGTTCTTCGCCTCCTGGT (SEQ ID NO: 8)

and

    5'TGGTGAGCCCAGAGAGTCTCTG (SEQ ID NO: 9).

The four individual pools from each of the positive master pools werethen screened by PCR using the same two primers, and positives wereverified by PCR with use of primers

    5'TGGAGTTCTTCGCCTCCTGGT (SEQ ID NO: 8)

and

    5'ATGATGGGACTCCACGGCGTCAAT (SEQ ID NO: 10).

One of the positive pools was plated, and colony lifts on nitrocellulosefilters were hybridized overnight in a hybridization solution (0.9MNaCl, 0.09M sodium citrate, 1% w/v Ficoll type 400, 1% w/vPolyvinylpyrrolidone, 1% w/v BSA, and 0.1% w/v SDS) at a temperature of54° C., with ³² P-labeled 5'GGGAAGCCAGGGATGTTGAAGTCT (SEQ ID NO: 11).Filters were washed with a washing solution (0.3M NaCl, 0.03M sodiumcitrate and 0.5% SDS), at room temperature for one hour, followed by afifteen-minute wash at 54° C., to remove unhybridized probe. Filterswere exposed to film overnight.

The following day, individual positive colonies were picked and screenedby PCR using as primers

    5'CGAAGAGTACCTGGCTCTGATC (SEQ ID NO: 12)

and

    5'TGGTGAGCCCAGAGAGTCTCTG (SEQ ID NO: 9).

The colonies were also screened with

    5'GGGAAGCCAGGGATGTTGAAGTCT (SEQ ID NO: 11),

and

    5'TGGAGTTCTTCGCCTCCTGGT (SEQ ID NO: 8).

Plasmid DNA from the positive clones was sequenced. Two cDNA clones wereisolated. The sequence of one of the clones included an open readingframe consisting of 1812 bases having the nucleic acid sequence shown inFIGS. 2A-2C (SEQ ID NO: 3), which encodes a protein of 604 amino acids,also shown in FIGS. 2A-2C (SEQ ID NO: 4), beginning with what is mostlikely a signal peptide. The second clone contained a large region ofsequence identity with the first clone, the 5' end of the second clonealigning with nucleotide 429 of the first clone. It is likely that thesecond clone was the result of 5'-end truncation that occurred in theprocess of cloning. The predicted open reading frame obtained byappending the sequence of clone 2 to nucleotides 1-428 of the openreading frame of clone 1 results in a molecule of 2103 nucleotides inlength, shown in FIGS. 3A-3D (SEQ ID NO: 5). This DNA sequence encodes aprotein of 701 amino acids in length, the predicted amino acid sequenceof which (SEQ ID NO: 6) is also shown in FIGS. 3A-3D.

Recombinant Expression of PDPFs

"293" cells (American Type Culture Collection, Rockville, Md., accessionno. CRL 1573), which are human embryonic kidney cells transfected withthe Ad5 adenovirus strain, were transfected with the large T antigen ofSV40 to give a cell line designated as "293T". These cells were platedin a fibronectin-coated six-well plate at 1×10⁶ cells/well. The cellswere then incubated overnight at a temperature of 37° C., under anatmosphere of 5% CO₂. Water and 5 μg of PDPF/pJT2 plasmid DNA (H₂O+DNA=90 μl) were combined, then 100 μl of 2× Hepes buffered saline wereadded to this mixture. The PDPF/pJT2 vector was constructed bysubcloning the cDNA for PDPF-1 or PDPF-2 polypeptide into expressionvector pJT2 (pJT2 is a known vector described in published PCTapplication WO 94/28133). The resulting mixture was sterile filteredwith a Costar spin-x filter. Then, 10 μl of 2.5 M CaCl₂ were added andmixed, and the mixture was permitted to stand at room temperature undera hood for thirty minutes. Air was bubbled into each DNA mixture (2 mlpipette+20 μl tip). One milliliter of media was removed from a well andcombined with the DNA mixture, then transferred to the 293T cells andthe cells were incubated overnight at 37° C., under 5% CO₂. The mediawere separated from the cells by aspiration and replaced with 2.5 ml ofIscove's Modified Dulbecco's Medium per well. The cells were incubatedfor three to four days at 37° C., under 5% CO₂. The conditioned mediawere harvested and the cellular debris was pelleted.

In Situ Hybridization Analysis of PDPF

These studies were performed for the detection of the presence of mRNAin various rat tissues of PDPF. For this analysis, a probe of about onekilobase prepared against the N-terminus of mouse PDPF was used. A panelof normal embryonic and adult rat tissues was fixed in 4%paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. Prior toin situ hybridization, tissues were permeabilized with 0.2M HCL,followed by digestion with Proteinase K and acetylation withtriethanolamine and acetic anhydride. Sections were hybridized with a ³³P-labeled riboprobe overnight at 55° C., then subjected to a highstringency wash in 0.1× SSC at 60° C. Slides were dipped in Kodak NTB2emulsion, exposed at 4° C. for two to three weeks, developed, and thencounterstained. Sections were examined with darkfield and standardillumination to allow simultaneous evaluation of tissue morphology andhybridization signal. The results are shown in FIGS. 4A-4G.

Overall, the N-terminal probe produced a relatively restricteddistribution of signal in the tissue sections from both embryo and adultrat, with little or no background signal. A majority of the signal inpositive tissues was localized to ductal, squamous or columnarepithelium. In embryonic tissue, strong signal (indicative of thepresence of significant amounts of mRNA for PDPF) was observed in thepancreas, the kidney collecting tubules, at thenasal-tracheal-esophageal junction, the heart, skin, and in theplacenta. A strong signal was also observed in several large vesselssuch as the hepatic and pulmonary arteries and in the endocardium of theheart, but it appeared to be absent from the microvasculature. Moderateor diffuse signal was detected in adrenal medullary cells, theintestine, ovary and testes. The brain, lungs, thymus, skeletal muscleand liver were negative. Some scattered cells of unknown origin wereobserved in developing bones, ribs, vertebrae and around jaw cartilage.In adult rat tissue, expression of mRNA for PDPF was strong in severalorgans. In the male reproductive system, there was intense signal in theepithelium of the testes and in the vesicular gland. There was alsostrong signal in the epididymus and prostate gland, and variable signalin the seminiferous tubules within the testis. Some developingspermatids also expressed some signal. There was also signal in thefemale reproductive system, including the germinal epithelium of growingand primordial follicles, but not in the corpus luteum of the ovaries,and some signal in the outermost layer of the stratified epithelium ofthe uterus. Breast ductal epithelium had intense signal. The stomach hada high level of signal in keratinized and glandular epithelium, as didthe epithelial keratinocytes of the esophagus and the skin. In the skin,the signal for PDPF was particularly intense in the cells adjacent tothe hair shaft (these cells are probably sebaceous cells). The strongsignal observed in embryonic pancreas was found to have decreased in theadult to a level that was only modestly above background. In the adultintestine, there was a variable level of signal in the epithelium thatwas strongest in the colon. In the lung, there was modest signal aroundsome large bronchi, and also some signal around some ducts in thesubmaxillary gland. Other tissues did not have strong specific signal,although several did have a grain density above background.

Human tumors from various sources were also screened with the PDPFprobe. Very strong signal was observed in two different breast and lungtumors that were sampled from metastatic sites in the brain. Anothermetastatic breast tumor also expressed a moderate signal for PDPF mRNA.

The expression of PDPF at the protein level was also examined in severalprostate carcinoma cell lines and in normal prostate epithelial cells.Results are shown in FIG. 5. These proteins were detected in theconditioned media of all three prostate carcinoma cell lines tested, butnot in normal prostate epithelial cells. The longer form (SEQ ID NO: 6)was expressed strongly in LNCaP and PC3 carcinoma cells, and weakly inDU145 cells. In contrast, the shorter form (SEQ ID NO: 4) was expressedin DU145 and PC3 carcinoma cells at a much lower level, and not at allin LNCaP cells. These data support the in vitro data (see below)suggesting that these proteins are associated with, and active on,prostate carcinoma cells. In addition, these results suggest that theproteins may function as autocrine growth factors in prostate carcinomacells. Expression was also analyzed in bone stromal tumor cell lines.Strong immunoreactive bands, corresponding to both the longer andshorter forms, were detected in the conditioned media of OHS4 and U20Sosteosarcoma cells. Expression in two other osteosarcoma cell lines,SaOs2 and TE85, was at a much lower level, and no expression could bedetected in MG63 osteosarcoma cells. These data confirm thatosteosarcoma cells, which are cells derived from the bone stroma,express and secrete PDPF.

Procedure for Cell Proliferation Assays

Cells were plated at 2-5×10³ cells/well into 96-well plates in 100 ml ofDMEM (GIBCO/BRL, Gaithersburg, Md.) or RPMI (GIBCO/BRL, Gaithersburg,Md.) containing 5% charcoal/dextran-stripped fetal bovine serum (GeminiBio-Products, Calabasas, Calif.), L-glutamine, non-essential amino acidsand penicillin/streptomycin. The cell cultures were allowed to attachand spread overnight. On the following day, the media was aspirated andreplaced by the assay media, DMEM or RPMI containing 2-5%charcoal/dextran-stripped fetal bovine serum, L-glutamine, non-essentialamino acids and penicillin/streptomycin, and the samples to be tested.Test samples consisted of 1× or 5× concentrated conditioned media (CM)from 293T cells that had been separately transfected with DNA for PDPF-1or PDPF-2 (see above). Western blots demonstrated that the transfected293T cell-conditioned media contained significant levels of PDPF at theexpected molecular weight for each isoform. As a negative control, 293Tcells were mock transfected (i.e., with non-PDPF containing vector),after which conditioned media were collected and tested in parallel.Western blots of negative control conditioned media showed no expressionof any PDPF. Conditioned media from PDPF or mock-transfected 293T cellswere added to triplicate wells for each data point. Additional controlsincluded cells incubated with assay media or growth media alone. Plateswere incubated for 3-4 days before cell proliferation was determinedusing the Celltiter 96 Aqueous non-radioactive cell proliferation assay(Promega, Madison, Wis.), and read on a Molecular Devices UVmax kineticmicroplate reader at 490 nm. Results for each data point were expressedas the mean from triplicate wells±standard deviation. PDPF was deemed tostimulate proliferation of a cell line if an increase of at least 20%over negative controls was observed.

Results of Cell Proliferation Assay

PDPF-1 (SEQ ID NO: 4), i.e., the "shorter" PDPF form, and PDPF-2 (SEQ IDNO: 6), i.e., the "longer" PDPF form, were evaluated for mitogenicactivity against various normal and abnormal (cancer) cell lines. Theresults of this evaluation are presented in Table 1 below. Designationsfor the cell lines tested are as follows: NbE 1.4 (normal rat prostateepithelial cells); LNCaP (human prostate carcinoma cells-lymph nodemetastasis); IEC 18 (normal rat ileum epithelial cells, i.e., smallintestine); HUVEC (human umbilical vein endothelial cells); SaOS2 (humanprimary osteogenic sarcoma cells); HEPM (human embryonal palatalmesenchyme cells); MiaPaCa (human pancreatic carcinoma cells); JAR(human placental choriocarcinoma cells); PC-3 (human prostateadenocarcinoma cells-bony metastasis); DU145 (human prostate carcinomacells-brain metastasis); Rat2 (normal rat embryonic fibroblasts); FADU(human pharynx squamous cell carcinoma cells); CAPAN I (human pancreaticadenocarcinoma cells--liver metastasis); CAPAN II (human pancreaticadenocarcinoma cells); and SW 480 (human colon adenocarcinoma cells). Asindicated above, proliferation was considered to have occurred if therewas an increase in cell population of at least twenty percent over thenegative controls. Within this criterion, the symbol "++" indicatesstrong proliferation, "+" indicates moderate proliferation, "--"indicates no proliferation, and "±" indicates marginal proliferation."N.D." indicates value was not determined.

                  TABLE 1                                                         ______________________________________                                        Cell Proliferation Assay                                                              1× CM       5× CM                                                   PDPF-1      PDPF-2      PDPF-1                                      Cell Line SEQ. ID NO: 4                                                                             SEQ. ID NO: 6                                                                             SEQ ID NO: 4                                ______________________________________                                        NbE 1.4   --          --          --                                          LNCaP     ++          +/-         +                                           IEC 18    +/-         N.D.        +/-                                         HUVEC     ++          N.D.        ++                                          SaOS2     +           --          --                                          HEPM      +           --          --                                          MiaPaCa   --          --          --                                          JAR       ++          ++          +/-                                         PC-3      --          --          --                                          DU145     +           +           --                                          Rat2      --          N.D.        --                                          FADU      ++          --          --                                          CAPAN I   --          N.D.        --                                          CAPAN II  --          N.D.        --                                          SW 480    --          N.D.        --                                          ______________________________________                                    

Useful Applications

Based on the foregoing results, it is expected that the PDPF proteins ofthis invention will be useful as growth factors to promote theproliferation, growth and survival of responsive cells tissues,including but not necessarily limited to prostate and epithelial cells.Because of the demonstrated effect of PDPF on prostate cancer cells invitro (above), this growth factor should prove especially useful inresearch studies for the improved understanding of the development andprogression of prostate cancer. For in vivo applications, thepolypeptides of this invention can be formulated into pharmaceuticallyacceptable compositions comprising an effective cell or tissue growthpromoting amount of the polypeptide and a pharmaceutically inertcarrier, excipient and/or diluent, optionally together with otherconventional ingredients.

The polypeptide growth factors of this invention are also expected to beutilizable in the identification and development of inhibitors ofPDPF-mediated tumor growth, as in vitro assays and test methods designedto detect any appreciable mediation of cell proliferation, growth orsurvival activity of the factors in the presence of other specificmolecules. Such inhibitors or "antagonists" of PDPF can serve asanti-tumor agents for the treatment of PDPF-stimulated tumor growth.

Antibodies specific to PDPF and nucleic acids such as DNA encoding fulllength or partial sequences of PDPF, as described herein, will be usefulin the diagnosis and treatment of diseases involving prostate orepithelial tumors. The antibodies of this invention can be usedtherapeutically, such as to inhibit binding of the polypeptide to itsreceptor. The antibodies can further be used for diagnostic purposes,such as in labeled form to detect the presence of the polypeptide in abody fluid, tissue sample or other extract. In one method, a biologicalsample comprising a tissue or fluid specimen is contacted with a labeled(radioactively or fluorescently) antibody specific to PDPF, and thelabeled reaction product of the antibody and PDPF is detected. Inanother method, a tissue or fluid specimen is contacted with a labeledprobe for a nucleic acid molecule that encodes PDPF, and the labeledhybridization product is detected.

Deposits of Microorganisms

The following recombinant plasmid DNA were deposited on Oct. 4, 1996,with the American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209. The first listed contains cDNA encoding PDPF-1(see nucleotide SEQ ID NO: 3), and the second (SEQ ID NO: 7).

    ______________________________________                                        Deposit Name        ATTC Accession No.                                        ______________________________________                                        pSPORT1: PDPF-1 p2N14.4                                                                           98194                                                     pGEM5: PDPF-2 cl.1  98195                                                     ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 12                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1617 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - ATGGAATCCA GGTCCTTCTA TACCGCTTAC CTGCAGAGAC TCTCTGGGCT CA - #CCAGGGAG         60                                                                          - GCTGCCCAGA CCACAGTTGC ACCAACCACT GCTAACAAGA TAGCTCCCAC TG - #TTTGGAAA        120                                                                          - TTGGCAGATC GCTCCAAGAT CTACATGGCT GACCTGGAAT CTGCACTGCA CT - #ACATCCTG        180                                                                          - CGGATAGAAG TGGGCAGGTT CCCGGTCCTG GAAGGGCAGC GCCTGGGTGG CC - #CTGAAAAA        240                                                                          - GTTTGTGGCA GTTCTGGCCA AGTATTTCCT GGGCGGCCCT TAGTCCAGAA CT - #TTCTGCAC        300                                                                          - TCCGTGAATG AATGGCTCAA GAGGCAGAAG AGAAATAAAA TTCCCTACAG TT - #TCTTTAAA        360                                                                          - ACTGCCCTGG ACGACAGGAA AGAGGGTGCC GTTCTTGCCA AGAAGGTGAA CT - #GGATTGGC        420                                                                          - TGCCAGGGGA GTGAGCCGCA TTTCCGGGGC TTTCCCTGCT CCTGTGGGTC CT - #CTTCCACT        480                                                                          - TCTAGACTGT GCAGGCAGCT CGGATCAAAA TGTAGACCAC TCACAGGAAG CA - #CGCAAGGC        540                                                                          - CAAGGAGGTC CTCCCAGCCA TCCGAGGCTA GCTGCACTAC TTCTTCGGCT GC - #CGAGACTG        600                                                                          - CGCAAGCCAC TTCGAGCAGA TGCTGCTGCC TCCATGCACC GGGTGGGGAG TC - #CCAACGCC        660                                                                          - GCTGTCCTCT GGCTCTGGTC TAGCCACAAC AGGGTCAATG CTCGCTTGCA GG - #TGCCCCCA        720                                                                          - GCGAGGACCC CCAGTTCCCC AAGGTGCAGT GGCCACCCCG TGAACTTTGT TC - #TGCCTGCC        780                                                                          - ACAATGAACG CCTGGATGTG CCCGTGTGGG ACGTGGAAGC CACCCTCAAC TT - #CCTCAAGG        840                                                                          - CCCACTTCTC CCCAAGCAAC ATCATCCTGG ACTTCCCTGC AGCTGGGTCA GC - #TGCCGGAG        900                                                                          - GGATGTGCAG AATGTGCAGC CGCCCCAGAG CTGGCGATGG GAGCCCTGGA GC - #TGGAAAGC        960                                                                          - CGGAATTCAA CTCTGGACCC TGGGAAGCCT GAGATGATGA AGTCCCCCAC AA - #ACACCACC       1020                                                                          - CCACATGTGC CGGCTGAGGG ACCTGAGGCA AGTCGACCCC CGAAGCTGCA CC - #CTGGCCTC       1080                                                                          - AGAGCTGCAC CAGGCCAGGA GCCTCCTGAG CACATGGCAG ACGTTCAGAG GA - #ATGAGCAG       1140                                                                          - GACGAGCCGC TTGGGCAGTG GCACTTACGA AGCGAGACAC AGGGGCTGCA TT - #GCTGGCTG       1200                                                                          - AGTCCAGGGC TGAGAAGAAC CGCCTCTGGG GCCCTTTGGA GGTCAGGCGC GT - #GGGCCGCA       1260                                                                          - GCTCCAAGCA GCTGGTCGAC ATCCCTGAGG CCAGCTGGAG GCCCGAGCTG GA - #CGGGCCGA       1320                                                                          - GGCCAGTGGC TGCAGGTGCT GGGAGGGGGC TTCTCTTACC TGGACATCAG CC - #TCTGTGTG       1380                                                                          - GGGCTCTATC CCTGTCCTTC ATGGGCCTGC TGGCATGTAC ACCTACTTCC AG - #GCCAAGAT       1440                                                                          - AAGGCCCTGA ACCGGATGCT GGCCACCCTG CAGCCTGAAC CACCTGGGGA GG - #AGGCGGGA       1500                                                                          - GAGGGAGCTG CCATCTCTAG GCACCTCAAG CCCCCTGACC CCATTCCCTC CC - #CTCCCACC       1560                                                                          - CCTTGCTCCT TGTCTGGCCT AGAAGTGTGG GAAATTCAGG AAAACGAGTT GC - #TCCAG          1617                                                                          - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 539 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Glu Ser Arg Ser Phe Tyr Thr Ala Tyr Le - #u Gln Arg Leu Ser Gly         #                15                                                           - Leu Thr Arg Glu Ala Ala Gln Thr Thr Val Al - #a Pro Thr Thr Ala Asn         #            30                                                               - Lys Ile Ala Pro Thr Val Trp Lys Leu Ala As - #p Arg Ser Lys Ile Tyr         #        45                                                                   - Met Ala Asp Leu Glu Ser Ala Leu His Tyr Il - #e Leu Arg Ile Glu Val         #    60                                                                       - Gly Arg Phe Pro Val Leu Glu Gly Gln Arg Le - #u Gly Gly Pro Glu Lys         #80                                                                           - Val Cys Gly Ser Ser Gly Gln Val Phe Pro Gl - #y Arg Pro Leu Val Gln         #                95                                                           - Asn Phe Leu His Ser Val Asn Glu Trp Leu Ly - #s Arg Gln Lys Arg Asn         #           110                                                               - Lys Ile Pro Tyr Ser Phe Phe Lys Thr Ala Le - #u Asp Asp Arg Lys Glu         #       125                                                                   - Gly Ala Val Leu Ala Lys Lys Val Asn Trp Il - #e Gly Cys Gln Gly Ser         #   140                                                                       - Glu Pro His Phe Arg Gly Phe Pro Cys Ser Cy - #s Gly Ser Ser Ser Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Arg Leu Cys Arg Gln Leu Gly Ser Lys Cy - #s Arg Pro Leu Thr Gly         #               175                                                           - Ser Thr Gln Gly Gln Gly Gly Pro Pro Ser Hi - #s Pro Arg Leu Ala Ala         #           190                                                               - Leu Leu Leu Arg Leu Pro Arg Leu Arg Lys Pr - #o Leu Arg Ala Asp Ala         #       205                                                                   - Ala Ala Ser Met His Arg Val Gly Ser Pro As - #n Ala Ala Val Leu Trp         #   220                                                                       - Leu Trp Ser Ser His Asn Arg Val Asn Ala Ar - #g Leu Gln Val Pro Pro         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ala Arg Thr Pro Ser Ser Pro Arg Cys Ser Gl - #y His Pro Val Asn Phe         #               255                                                           - Val Leu Pro Ala Thr Met Asn Ala Trp Met Cy - #s Pro Cys Gly Thr Trp         #           270                                                               - Lys Pro Pro Ser Thr Ser Ser Arg Pro Thr Se - #r Pro Gln Ala Thr Ser         #       285                                                                   - Ser Trp Thr Ser Leu Gln Leu Gly Gln Leu Pr - #o Glu Gly Cys Ala Glu         #   300                                                                       - Cys Ala Ala Ala Pro Glu Leu Ala Met Gly Al - #a Leu Glu Leu Glu Ser         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Arg Asn Ser Thr Leu Asp Pro Gly Lys Pro Gl - #u Met Met Lys Ser Pro         #               335                                                           - Thr Asn Thr Thr Pro His Val Pro Ala Glu Gl - #y Pro Glu Ala Ser Arg         #           350                                                               - Pro Pro Lys Leu His Pro Gly Leu Arg Ala Al - #a Pro Gly Gln Glu Pro         #       365                                                                   - Pro Glu His Met Ala Asp Val Gln Arg Asn Gl - #u Gln Asp Glu Pro Leu         #   380                                                                       - Gly Gln Trp His Leu Arg Ser Glu Thr Gln Gl - #y Leu His Cys Trp Leu         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Ser Pro Gly Leu Arg Arg Thr Ala Ser Gly Al - #a Leu Trp Arg Ser Gly         #               415                                                           - Ala Trp Ala Ala Ala Pro Ser Ser Trp Ser Th - #r Ser Leu Arg Pro Ala         #           430                                                               - Gly Gly Pro Ser Trp Thr Gly Arg Gly Gln Tr - #p Leu Gln Val Leu Gly         #       445                                                                   - Gly Gly Phe Ser Tyr Leu Asp Ile Ser Leu Cy - #s Val Gly Leu Tyr Pro         #   460                                                                       - Cys Pro Ser Trp Ala Cys Trp His Val His Le - #u Leu Pro Gly Gln Asp         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Lys Ala Leu Asn Arg Met Leu Ala Thr Leu Gl - #n Pro Glu Pro Pro Gly         #               495                                                           - Glu Glu Ala Gly Glu Gly Ala Ala Ile Ser Ar - #g His Leu Lys Pro Pro         #           510                                                               - Asp Pro Ile Pro Ser Pro Pro Thr Pro Cys Se - #r Leu Ser Gly Leu Glu         #       525                                                                   - Val Trp Glu Ile Gln Glu Asn Glu Leu Leu Gl - #n                             #   535                                                                       - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1812 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - ATGAGGAGGT GCAACAGCGG CTCCGGGCCG CCGCCGTCGC TGCTGCTGCT GC - #TGCTGTGG         60                                                                          - CTGCTCGCGG TTCCCGGCGC TAACGCGGCC CCGCGGTCGG CGCTCTATTC GC - #CTTCCGAC        120                                                                          - CCGCTGACGC TGCTGCAGGC GGACACGGTG CGCGGCGCGG TGCTGGGCTC CC - #GCAGCGCC        180                                                                          - TGGGCCGTGG AGTTCTTCGC CTCCTGGTGC GGCCACTGCA TCGCCTTCGC CC - #CGACGTGG        240                                                                          - AAGGCGCTGG CCGAAGACGT CAAAGCCTGG AGGCCGGCCC TGTATCTCGC CG - #CCCTGGAC        300                                                                          - TGTGCTGAGG AGACCAACAG TGCAGTCTGC AGAGACTTCA ACATCCCTGG CT - #TCCCGACT        360                                                                          - GTGAGGTTCT TCAAGGCCTT TACCAAGAAC GGCTCGGGAG CAGTATTTCC AG - #TGGCTGGT        420                                                                          - GCTGACGTGC AGACGCTGCG GGAGAGGCTC ATTGACGCCC TGGAGTCCCA TC - #ATGACACG        480                                                                          - TGGCCCCCAG CCTGTCCCCC ACTGGAGCCT GCCAAGCTGG AGGAGATTGA TG - #GATTCTTT        540                                                                          - GCGAGAAATA ACGAAGAGTA CCTGGCTCTG ATCTTTGAAA AGGGAGGCTC CT - #ACCTGGGT        600                                                                          - AGAGAGGTGG CTCTGGACCT GTCCCAGCAC AAAGGCGTGG CGGTGCGCAG GG - #TGCTGAAC        660                                                                          - ACAGAGGCCA ATGTGGTGAG AAAGTTTGGT GTCACCGACT TCCCCTCTTG CT - #ACCTGCTG        720                                                                          - TTCCGGAATG GCTCTGTCTC CCGAGTCCCC GTGCTCATGG AATCCAGGTC CT - #TCTATACC        780                                                                          - GCTTACCTGC AGAGACTCTC TGGGCTCACC AGGGAGGCTG CCCAGACCAC AG - #TTGCACCA        840                                                                          - ACCACTGCTA ACAAGATAGC TCCCACTGTT TGGAAATTGG CAGATCGCTC CA - #AGATCTAC        900                                                                          - ATGGCTGACC TGGAATCTGC ACTGCACTAC ATCCTGCGGA TAGAAGTGGG CA - #GGTTCCCG        960                                                                          - GTCCTGGAAG GGCAGCGCCT GGTGGCCCTG AAAAAGTTTG TGGCAGTGCT GG - #CCAAGTAT       1020                                                                          - TTCCCTGGCC GGCCCTTAGT CCAGAACTTC CTGCACTCCG TGAATGAATG GC - #TCAAGAGG       1080                                                                          - CAGAAGAGAA ATAAAATTCC CTACAGTTTC TTTAAAACTG CCCTGGACGA CA - #GGAAAGAG       1140                                                                          - GGTGCCGTTC TTGCCAAGAA GGTGAACTGG ATTGGCTGCC AGGGGAGTGA GC - #CGCATTTC       1200                                                                          - CGGGGCTTTC CCTGCTCCCT GTGGGTCCTC TTCCACTTCT TGACTGTGCA GG - #CAGCTCGC       1260                                                                          - CAAAATGTAG ACCACTCACA GGAAGCAGCC AAGGCCAAGG AGGTCCTCCC AG - #CCATCCGA       1320                                                                          - GGCTACGTGC ACTACTTCTT CGGCTGCCGA GACTGCGCTA GCCACTTCGA GC - #AGATGGCT       1380                                                                          - GCTGCCTCCA TGCACCGGGT GGGGAGTCCC AACGCCGCTG TCCTCTGGCT CT - #GGTCTAGC       1440                                                                          - CACAACAGGG TCAATGCTCG CCTTGCAGGT GCCCCCAGCG AGGACCCCCA GT - #TCCCCAAG       1500                                                                          - GTGCAGTGGC CACCCCGTGA ACTTTGTTCT GCCTGCCACA ATGAACGCCT GG - #ATGTGCCC       1560                                                                          - GTGTGGGACG TGGAAGCCAC CCTCAACTTC CTCAAGGCCC ACTTCTCCCC AA - #GCAACATC       1620                                                                          - ATCCTGGACT TCCCTGCAGC TGGGTCAGCT GCCCGGAGGG ATGTGCAGAA TG - #TGGCAGCC       1680                                                                          - GCCCCAGAGC TGGCGATGGG AGCCCTGGAG CTGGAAAGCC GGAATTCAAC TC - #TGGACCCT       1740                                                                          - GGGAAGCCTG AGATGATGAA GTCCCCCACA AACACCACCC CACATGTGCC GG - #CTGAGGGA       1800                                                                          #     1812                                                                    - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 604 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1812                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Arg Arg Cys Asn Ser Gly Ser Gly Pro Pr - #o Pro Ser Leu Leu Leu         #                15                                                           - Leu Leu Leu Trp Leu Leu Ala Val Pro Gly Al - #a Asn Ala Ala Pro Arg         #            30                                                               - Ser Ala Leu Tyr Ser Pro Ser Asp Pro Leu Th - #r Leu Leu Gln Ala Asp         #        45                                                                   - Thr Val Arg Gly Ala Val Leu Gly Ser Arg Se - #r Ala Trp Ala Val Glu         #    60                                                                       - Phe Phe Ala Ser Trp Cys Gly His Cys Ile Al - #a Phe Ala Pro Thr Trp         #80                                                                           - Lys Ala Leu Ala Glu Asp Val Lys Ala Trp Ar - #g Pro Ala Leu Tyr Leu         #                95                                                           - Ala Ala Leu Asp Cys Ala Glu Glu Thr Asn Se - #r Ala Val Cys Arg Asp         #           110                                                               - Phe Asn Ile Pro Gly Phe Pro Thr Val Arg Ph - #e Phe Lys Ala Phe Thr         #       125                                                                   - Lys Asn Gly Ser Gly Ala Val Phe Pro Val Al - #a Gly Ala Asp Val Gln         #   140                                                                       - Thr Leu Arg Glu Arg Leu Ile Asp Ala Leu Gl - #u Ser His His Asp Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Trp Pro Pro Ala Cys Pro Pro Leu Glu Pro Al - #a Lys Leu Glu Glu Ile         #               175                                                           - Asp Gly Phe Phe Ala Arg Asn Asn Glu Glu Ty - #r Leu Ala Leu Ile Phe         #           190                                                               - Glu Lys Gly Gly Ser Tyr Leu Gly Arg Glu Va - #l Ala Leu Asp Leu Ser         #       205                                                                   - Gln His Lys Gly Val Ala Val Arg Arg Val Le - #u Asn Thr Glu Ala Asn         #   220                                                                       - Val Val Arg Lys Phe Gly Val Thr Asp Phe Pr - #o Ser Cys Tyr Leu Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Phe Arg Asn Gly Ser Val Ser Arg Val Pro Va - #l Leu Met Glu Ser Arg         #               255                                                           - Ser Phe Tyr Thr Ala Tyr Leu Gln Arg Leu Se - #r Gly Leu Thr Arg Glu         #           270                                                               - Ala Ala Gln Thr Thr Val Ala Pro Thr Thr Al - #a Asn Lys Ile Ala Pro         #       285                                                                   - Thr Val Trp Lys Leu Ala Asp Arg Ser Lys Il - #e Tyr Met Ala Asp Leu         #   300                                                                       - Glu Ser Ala Leu His Tyr Ile Leu Arg Ile Gl - #u Val Gly Arg Phe Pro         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Val Leu Glu Gly Gln Arg Leu Val Ala Leu Ly - #s Lys Phe Val Ala Val         #               335                                                           - Leu Ala Lys Tyr Phe Pro Gly Arg Pro Leu Va - #l Gln Asn Phe Leu His         #           350                                                               - Ser Val Asn Glu Trp Leu Lys Arg Gln Lys Ar - #g Asn Lys Ile Pro Tyr         #       365                                                                   - Ser Phe Phe Lys Thr Ala Leu Asp Asp Arg Ly - #s Glu Gly Ala Val Leu         #   380                                                                       - Ala Lys Lys Val Asn Trp Ile Gly Cys Gln Gl - #y Ser Glu Pro His Phe         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Arg Gly Phe Pro Cys Ser Leu Trp Val Leu Ph - #e His Phe Leu Thr Val         #               415                                                           - Gln Ala Ala Arg Gln Asn Val Asp His Ser Gl - #n Glu Ala Ala Lys Ala         #           430                                                               - Lys Glu Val Leu Pro Ala Ile Arg Gly Tyr Va - #l His Tyr Phe Phe Gly         #       445                                                                   - Cys Arg Asp Cys Ala Ser His Phe Glu Gln Me - #t Ala Ala Ala Ser Met         #   460                                                                       - His Arg Val Gly Ser Pro Asn Ala Ala Val Le - #u Trp Leu Trp Ser Ser         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - His Asn Arg Val Asn Ala Arg Leu Ala Gly Al - #a Pro Ser Glu Asp Pro         #               495                                                           - Gln Phe Pro Lys Val Gln Trp Pro Pro Arg Gl - #u Leu Cys Ser Ala Cys         #           510                                                               - His Asn Glu Arg Leu Asp Val Pro Val Trp As - #p Val Glu Ala Thr Leu         #       525                                                                   - Asn Phe Leu Lys Ala His Phe Ser Pro Ser As - #n Ile Ile Leu Asp Phe         #   540                                                                       - Pro Ala Ala Gly Ser Ala Ala Arg Arg Asp Va - #l Gln Asn Val Ala Ala         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ala Pro Glu Leu Ala Met Gly Ala Leu Glu Le - #u Glu Ser Arg Asn Ser         #               575                                                           - Thr Leu Asp Pro Gly Lys Pro Glu Met Met Ly - #s Ser Pro Thr Asn Thr         #           590                                                               - Thr Pro His Val Pro Ala Glu Gly Pro Glu Le - #u Ile                         #       600                                                                   - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2103 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - ATGAGGAGGT GCAACAGCGG CTCCGGGCCG CCGCCGTCGC TGCTGCTGCT GC - #TGCTGTGG         60                                                                          - CTGCTCGCGG TTCCCGGCGC TAACGCGGCC CCGCGGTCGG CGCTCTATTC GC - #CTTCCGAC        120                                                                          - CCGCTGACGC TGCTGCAGGC GGACACGGTG CGCGGCGCGG TGCTGGGCTC CC - #GCAGCGCC        180                                                                          - TGGGCCGTGG AGTTCTTCGC CTCCTGGTGC GGCCACTGCA TCGCCTTCGC CC - #CGACGTGG        240                                                                          - AAGGCGCTGG CCGAAGACGT CAAAGCCTGG AGGCCGGCCC TGTATCTCGC CG - #CCCTGGAC        300                                                                          - TGTGCTGAGG AGACCAACAG TGCAGTCTGC AGAGACTTCA ACATCCCTGG CT - #TCCCGACT        360                                                                          - GTGAGGTTCT TCAAGGCCTT TACCAAGAAC GGCTCGGGAG CAGTATTTCC AG - #TGGCTGGT        420                                                                          - GCTGACGTGC AGACGCTGCG GGAGAGGCTC ATTGACGCCC TGGAGTCCCA TC - #ATGACACG        480                                                                          - TGGCCCCCAG CCTGTCCCCC ACTGGAGCCT GCCAAGCTGG AGGAGATTGA TG - #GATTCTTT        540                                                                          - GCGAGAAATA ACGAAGAGTA CCTGGCTCTG ATCTTTGAAA AGGGAGGCTC CT - #ACCTGGGT        600                                                                          - AGAGAGGTGG CTCTGGACCT GTCCCAGCAC AAAGGCGTGG CGGTGCGCAG GG - #TGCTGAAC        660                                                                          - ACAGAGGCCA ATGTGGTGAG AAAGTTTGGT GTCACCGACT TCCCCTCTTG CT - #ACCTGCTG        720                                                                          - TTCCGGAATG GCTCTGTCTC CCGAGTCCCC GTGCTCATGG AATCCAGGTC CT - #TCTATACC        780                                                                          - GCTTACCTGC AGAGACTCTC TGGGCTCACC AGGGAGGCTG CCCAGACCAC AG - #TTGCACCA        840                                                                          - ACCACTGCTA ACAAGATAGC TCCCACTGTT TGGAAATTGG CAGATCGCTC CA - #AGATCTAC        900                                                                          - ATGGCTGACC TGGAATCTGC ACTGCACTAC ATCCTGCGGA TAGAAGTGGG CA - #GGTTCCCG        960                                                                          - GTCCTGGAAG GGCAGCGCCT GGTGGCCCTG AAAAAGTTTG TGGCAGTGCT GG - #CCAAGTAT       1020                                                                          - TTCCCTGGCC GGCCCTTAGT CCAGAACTTC CTGCACTCCG TGAATGAATG GC - #TCAAGAGG       1080                                                                          - CAGAAGAGAA ATAAAATTCC CTACAGTTTC TTTAAAACTG CCCTGGACGA CA - #GGAAAGAG       1140                                                                          - GGTGCCGTTC TTGCCAAGAA GGTGAACTGG ATTGGCTGCC AGGGGAGTGA GC - #CGCATTTC       1200                                                                          - CGGGGCTTTC CCTGCTCCCT GTGGGTCCTC TTCCACTTCT TGACTGTGCA GG - #CAGCTCGC       1260                                                                          - CAAAATGTAG ACCACTCACA GGAAGCAGCC AAGGCCAAGG AGGTCCTCCC AG - #CCATCCGA       1320                                                                          - GGCTACGTGC ACTACTTCTT CGGCTGCCGA GACTGCGCTA GCCACTTCGA GC - #AGATGGCT       1380                                                                          - GCTGCCTCCA TGCACCGGGT GGGGAGTCCC AACGCCGCTG TCCTCTGGCT CT - #GGTCTAGC       1440                                                                          - CACAACAGGG TCAATGCTCG CCTTGCAGGT GCCCCCAGCG AGGACCCCCA GT - #TCCCCAAG       1500                                                                          - GTGCAGTGGC CACCCCGTGA ACTTTGTTCT GCCTGCCACA ATGAACGCCT GG - #ATGTGCCC       1560                                                                          - GTGTGGGACG TGGAAGCCAC CCTCAACTTC CTCAAGGCCC ACTTCTCCCC AA - #GCAACATC       1620                                                                          - ATCCTGGACT TCCCTGCAGC TGGGTCAGCT GCCCGGAGGG ATGTGCAGAA TG - #TGGCAGCC       1680                                                                          - GCCCCAGAGC TGGCGATGGG AGCCCTGGAG CTGGAAAGCC GGAATTCAAC TC - #TGGACCCT       1740                                                                          - GGGAAGCCTG AGATGATGAA GTCCCCCACA AACACCACCC CACATGTGCC GG - #CTGAGGGA       1800                                                                          - CCTGAGGCAA GTCGACCCCC GAAGCTGCAC CCTGGCCTCA GAGCTGCACC AG - #GCCAGGAG       1860                                                                          - CCTCCTGAGC ACATGGCAGA GCTTCAGAGG AATGAGCAGG AGCAGCCGCT TG - #GGCAGTGG       1920                                                                          - CACTTGAGCA AGCGAGACAC AGGGGCTGCA TTGCTGGCTG AGTCCAGGGC TG - #AGAAGAAC       1980                                                                          - CGCCTCTGGG GCCCTTTGGA GGTCAGGCGC GTGGGCCGCA GCTCCAAGCA GC - #TGGTCGAC       2040                                                                          - ATCCCTGAGG GCCAGCTGGA GGCCCGAGCT GGACGGGGCC GAGGCCAGTG GC - #TGCAGCTT       2100                                                                          #           2103                                                              - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 701 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Met Arg Arg Cys Asn Ser Gly Ser Gly Pro Pr - #o Pro Ser Leu Leu Leu         #                15                                                           - Leu Leu Leu Trp Leu Leu Ala Val Pro Gly Al - #a Asn Ala Ala Pro Arg         #            30                                                               - Ser Ala Leu Tyr Ser Pro Ser Asp Pro Leu Th - #r Leu Leu Gln Ala Asp         #        45                                                                   - Thr Val Arg Gly Ala Val Leu Gly Ser Arg Se - #r Ala Trp Ala Val Glu         #    60                                                                       - Phe Phe Ala Ser Trp Cys Gly His Cys Ile Al - #a Phe Ala Pro Thr Trp         #80                                                                           - Lys Ala Leu Ala Glu Asp Val Lys Ala Trp Ar - #g Pro Ala Leu Tyr Leu         #                95                                                           - Ala Ala Leu Asp Cys Ala Glu Glu Thr Asn Se - #r Ala Val Cys Arg Asp         #           110                                                               - Phe Asn Ile Pro Gly Phe Pro Thr Val Arg Ph - #e Phe Lys Ala Phe Thr         #       125                                                                   - Lys Asn Gly Ser Gly Ala Val Phe Pro Val Al - #a Gly Ala Asp Val Gln         #   140                                                                       - Thr Leu Arg Glu Arg Leu Ile Asp Ala Leu Gl - #u Ser His His Asp Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Trp Pro Pro Ala Cys Pro Pro Leu Glu Pro Al - #a Lys Leu Glu Glu Ile         #               175                                                           - Asp Gly Phe Phe Ala Arg Asn Asn Glu Glu Ty - #r Leu Ala Leu Ile Phe         #           190                                                               - Glu Lys Gly Gly Ser Tyr Leu Gly Arg Glu Va - #l Ala Leu Asp Leu Ser         #       205                                                                   - Gln His Lys Gly Val Ala Val Arg Arg Val Le - #u Asn Thr Glu Ala Asn         #   220                                                                       - Val Val Arg Lys Phe Gly Val Thr Asp Phe Pr - #o Ser Cys Tyr Leu Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Phe Arg Asn Gly Ser Val Ser Arg Val Pro Va - #l Leu Met Glu Ser Arg         #               255                                                           - Ser Phe Tyr Thr Ala Tyr Leu Gln Arg Leu Se - #r Gly Leu Thr Arg Glu         #           270                                                               - Ala Ala Gln Thr Thr Val Ala Pro Thr Thr Al - #a Asn Lys Ile Ala Pro         #       285                                                                   - Thr Val Trp Lys Leu Ala Asp Arg Ser Lys Il - #e Tyr Met Ala Asp Leu         #   300                                                                       - Glu Ser Ala Leu His Tyr Ile Leu Arg Ile Gl - #u Val Gly Arg Phe Pro         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Val Leu Glu Gly Gln Arg Leu Val Ala Leu Ly - #s Lys Phe Val Ala Val         #               335                                                           - Leu Ala Lys Tyr Phe Pro Gly Arg Pro Leu Va - #l Gln Asn Phe Leu His         #           350                                                               - Ser Val Asn Glu Trp Leu Lys Arg Gln Lys Ar - #g Asn Lys Ile Pro Tyr         #       365                                                                   - Ser Phe Phe Lys Thr Ala Leu Asp Asp Arg Ly - #s Glu Gly Ala Val Leu         #   380                                                                       - Ala Lys Lys Val Asn Trp Ile Gly Cys Gln Gl - #y Ser Glu Pro His Phe         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Arg Gly Phe Pro Cys Ser Leu Trp Val Leu Ph - #e His Phe Leu Thr Val         #               415                                                           - Gln Ala Ala Arg Gln Asn Val Asp His Ser Gl - #n Glu Ala Ala Lys Ala         #           430                                                               - Lys Glu Val Leu Pro Ala Ile Arg Gly Tyr Va - #l His Tyr Phe Phe Gly         #       445                                                                   - Cys Arg Asp Cys Ala Ser His Phe Glu Gln Me - #t Ala Ala Ala Ser Met         #   460                                                                       - His Arg Val Gly Ser Pro Asn Ala Ala Val Le - #u Trp Leu Trp Ser Ser         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - His Asn Arg Val Asn Ala Arg Leu Ala Gly Al - #a Pro Ser Glu Asp Pro         #               495                                                           - Gln Phe Pro Lys Val Gln Trp Pro Pro Arg Gl - #u Leu Cys Ser Ala Cys         #           510                                                               - His Asn Glu Arg Leu Asp Val Pro Val Trp As - #p Val Glu Ala Thr Leu         #       525                                                                   - Asn Phe Leu Lys Ala His Phe Ser Pro Ser As - #n Ile Ile Leu Asp Phe         #   540                                                                       - Pro Ala Ala Gly Ser Ala Ala Arg Arg Asp Va - #l Gln Asn Val Ala Ala         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ala Pro Glu Leu Ala Met Gly Ala Leu Glu Le - #u Glu Ser Arg Asn Ser         #               575                                                           - Thr Leu Asp Pro Gly Lys Pro Glu Met Met Ly - #s Ser Pro Thr Asn Thr         #           590                                                               - Thr Pro His Val Pro Ala Glu Gly Pro Glu Al - #a Ser Arg Pro Pro Lys         #       605                                                                   - Leu His Pro Gly Leu Arg Ala Ala Pro Gly Gl - #n Glu Pro Pro Glu His         #   620                                                                       - Met Ala Glu Leu Gln Arg Asn Glu Gln Glu Gl - #n Pro Leu Gly Gln Trp         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - His Leu Ser Lys Arg Asp Thr Gly Ala Ala Le - #u Leu Ala Glu Ser Arg         #               655                                                           - Ala Glu Lys Asn Arg Leu Trp Gly Pro Leu Gl - #u Val Arg Arg Val Gly         #           670                                                               - Arg Ser Ser Lys Gln Leu Val Asp Ile Pro Gl - #u Gly Gln Leu Glu Ala         #       685                                                                   - Arg Ala Gly Arg Gly Arg Gly Gln Trp Leu Gl - #n Leu Ile                     #   700                                                                       - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2103 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - ATGAGGAGGT GCAACAGCGG CTCCGGGCCG CCGCCGTCGC TGCTGCTGCT GC - #TGCTGTGG         60                                                                          - CTGCTCGCGG TTCCCGGCGC TAACGCGGCC CCGCGGTCGG CGCTCTATTC GC - #CTTCCGAC        120                                                                          - CCGCTGACGC TGCTGCAGGC GGACACGGTG CGCGGCGCGG TGCTGGGCTC CC - #GCAGCGCC        180                                                                          - TGGGCCGTGG AGTTCTTCGC CTCCTGGTGC GGCCACTGCA TCGCCTTCGC CC - #CGACGTGG        240                                                                          - AAGGCGCTGG CCGAAGACGT CAAAGCCTGG AGGCCGGCCC TGTATCTCGC CG - #CCCTGGAC        300                                                                          - TGTGCTGAGG AGACCAACAG TGCAGTCTGC AGAGACTTCA ACATCCCTGG CT - #TCCCGACT        360                                                                          - GTGAGGTTCT TCAAGGCCTT TACCAAGAAC GGCTCGGGAG CAGTATTTCC AG - #TGGCTGGT        420                                                                          - GCTGACGTGC AGACGCTGCG GGAGAGGCTC ATTGACGCCC TGGAGTCCCA TC - #ATGACACG        480                                                                          - TGGCCCCCAG CCTGTCCCCC ACTGGAGCCT GCCAAGCTGG AGGAGATTGA TG - #GATTCTTT        540                                                                          - GCGAGAAATA ACGAAGAGTA CCTGGCTCTG ATCTTTGAAA AGGGAGGCTC CT - #ACCTGGGT        600                                                                          - AGAGAGGTGG CTCTGGACCT GTCCCAGCAC AAAGGCGTGG CGGTGCGCAG GG - #TGCTGAAC        660                                                                          - ACAGAGGCCA ATGTGGTGAG AAAGTTTGGT GTCACCGACT TCCCCTCTTG CT - #ACCTGCTG        720                                                                          - TTCCGGAATG GCTCTGTCTC CCGAGTCCCC GTGCTCATGG AATCCAGGTC CT - #TCTATACC        780                                                                          - GCTTACCTGC AGAGACTCTC TGGGCTCACC AGGGAGGCTG CCCAGACCAC AG - #TTGCACCA        840                                                                          - ACCACTGCTA ACAAGATAGC TCCCACTGTT TGGAAATTGG CAGATCGCTC CA - #AGATCTAC        900                                                                          - ATGGCTGACC TGGAATCTGC ACTGCACTAC ATCCTGCGGA TAGAAGTGGG CA - #GGTTCCCG        960                                                                          - GTCCTGGAAG GGCAGCGCCT GGTGGCCCTG AAAAAGTTTG TGGCAGTGCT GG - #CCAAGTAT       1020                                                                          - TTCCCTGGCC GGCCCTTAGT CCAGAACTTC CTGCACTCCG TGAATGAATG GC - #TCAAGAGG       1080                                                                          - CAGAAGAGAA ATAAAATTCC CTACAGTTTC TTTAAAACTG CCCTGGACGA CA - #GGAAAGAG       1140                                                                          - GGTGCCGTTC TTGCCAAGAA GGTGAACTGG ATTGGCTGCC AGGGGAGTGA GC - #CGCATTTC       1200                                                                          - GGGGGCTTTC CCTGCTCCCT GTGGGTCCTC TTCCACTTCT TGACTGTGCA GG - #CAGCTCGG       1260                                                                          - CAAAATGTAG ACCACTCACA GGAAGCAGCC AAGGCCAAGG AGGTCCTCCC AG - #CCATCCGA       1320                                                                          - GGCTACGTGC ACTACTTCTT CGGCTGCCGA GACTGCGCTA GCCACTTCGA GC - #AGATGGCT       1380                                                                          - GCTGCCTCCA TGCACCGGGT GGGGAGTCCC AACGCCGCTG TCCTCTGGCT CT - #GGTCTAGC       1440                                                                          - CACAACAGGG TCAATGCTCG CCTTGCAGGT GCCCCCAGCG AGGACCCCCA GT - #TCCCCAAG       1500                                                                          - GTGCAGTGGC CACCCCGTGA ACTTTGTTCT GCCTGCCACA ATGAACGCCT GG - #ATGTGCCC       1560                                                                          - GTGTGGGACG TGGAAGCCAC CCTCAACTTC CTCAAGGCCC ACTTCTCCCC AA - #GCAACATC       1620                                                                          - ATCCTGGACT TCCCTGCAGC TGGGTCAGCT GCCCGGAGGG ATGTGCAGAA TG - #TGGCAGCC       1680                                                                          - GCCCCAGAGC TGGCGATGGG AGCCCTGGAG CTGGAAAGCC GGAATTCAAC TC - #TGGACCCT       1740                                                                          - GGGAAGCCTG AGATGATGAA GTCCCCCACA AACACCACCC CACATGTGCC GG - #CTGAGGGA       1800                                                                          - CCTGAGGCAA GTCGACCCCC GAAGCTGCAC CCTGGCCTCA GAGCTGCACC AG - #GCCAGGAG       1860                                                                          - CCTCCTGAGC ACATGGCAGA GCTTCAGAGG AATGAGCAGG AGCAGCCGCT TG - #GGCAGTGG       1920                                                                          - CACTTGAGCA AGCGAGACAC AGGGGCTGCA TTGCTGGCTG AGTCCAGGGC TG - #AGAAGAAC       1980                                                                          - CGCCTCTGGG GCCCTTTGGA GGTCAGGCGC GTGGGCCGCA GCTCCAAGCA GC - #TGGTCGAC       2040                                                                          - ATCCCTGAGG GCCAGCTGGA GGCCCGAGCT GGACGGGGCC GAGGCCAGTG GC - #TGCAGCTT       2100                                                                          #           2103                                                              - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #21                CTGG T                                                     - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #                 22CTC TG                                                    - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #                24GCGT CAAT                                                  - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #                24TGAA GTCT                                                  - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #                 22TGA TC                                                    __________________________________________________________________________

What is claimed is:
 1. A method for promoting the growth andproliferation of cells comprising contacting such cells which areresponsive thereto with an effective amount of a placental-derivedprostate growth factor having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 4 and SEQ ID NO:
 6. 2. A method accordingto claim 1 in which the cells are prostate cells.
 3. A method accordingto claim 1 in which the cells are epithelial cells.
 4. A methodaccording to claim 1 which is carried out in vitro.
 5. A methodaccording to claim 1 which is carried out in vivo.